家蚕组织蛋白酶L（BmCathepsin L）基因鉴定、表达及其功能分析

doi:10.3864/j.issn.0578-1752.2017.16.018

摘要/Abstract

摘要： 【目的】鉴定克隆家蚕组织蛋白酶L基因BmCathepsin L（BmCat L），分析其序列和表达特征。分析家蚕组织蛋白酶L在大肠杆菌诱导下mRNA表达量的变化趋势，为进一步研究家蚕组织蛋白酶L的功能打下基础。【方法】基于家蚕基因组数据库（SilkDB）中序列BGIBMGA006893设计引物，利用RACE技术克隆家蚕组织蛋白酶L基因的cDNA全长序列，通过在线工具对基因结构和蛋白质分子量及等电点进行预测；在NCBI数据库下载其他物种组织蛋白酶L同源序列，利用Clustalx（1.83）和MEGA 6.0 软件进行同源比对并构建进化树。运用RT-PCR和qRT-PCR对其时空表达特征进行分析。选取该基因的特异性片段，经PCR扩增，测序验证后将其连接至pET-28载体，转化至Rosseta感受态表达菌株，经IPTG在适宜温度条件下诱导，获得组织蛋白酶L重组蛋白。然后再利用镍离子亲和层析法对该蛋白进行纯化，获得纯度较高的重组蛋白。最后利用大肠杆菌对家蚕血液系统进行攻毒，检测家蚕组织蛋白酶L基因的表达水平。【结果】通过查询家蚕基因组数据库和生物信息学分析得出，家蚕组织蛋白酶L基因定位于家蚕第10号染色体上，基因编号为BGIBMGA006893, nscaf2860。基因开放阅读框全长687 bp，编码228个氨基酸，含有保守的Pept_C1结构域。通过在线网站预测，得出该蛋白酶的分子量为26.132 kD，等电点为4.57。进化分析表明该基因与同为鳞翅目昆虫的草地贪夜蛾和黑脉金斑蝶亲缘关系最为接近。RT-PCR显示该基因集中表达于血细胞，在血液不同龄期也较高表达。用His抗体检测经纯化后的蛋白，最终成功孵育出纯化后的蛋白条带;大肠杆菌个体攻毒免疫试验检测显示，其mRNA表达水平随攻毒时间呈现抛物线式的显著性变化规律，从而揭示出BmCat L与家蚕免疫系统的关联性。【结论】克隆获得家蚕组织蛋白酶L基因的cDNA序列，发现其特异性高表达于血细胞。通过原核表达和蛋白纯化成功获得了该基因的重组蛋白。大肠杆菌的刺激能够显著上调其表达，推测组织蛋白酶L可能参与家蚕免疫反应。

关键词: 组织蛋白酶L基因, 家蚕, 基因克隆, 原核表达, 蛋白纯化, 免疫应激

Abstract: 【Objective】 The objective of this study is to identify and clone the BmCathepsin L (BmCat L) from silkworm (Bombyx mori), investigate its sequence features and expression profiles, and to clarify the response of BmCat L under E. coli stimulate in B. mori. Results of the study will provide a theoretical basis for further studying the function of BmCat L in B. mori.【Method】The primers was designed based on the sequence of BGIBMGA006893 in SilkDB database. The full length cDNA of BmCat L was acquired by RACE (rapid-amplification of cDNA ends) technology. The protein structure and molecular weight was predicted online. About the homologous sequences of Cathepsin L from other species were downloaded from NCBI, and the deduced amino acid sequence of putative BmCat L was aligned using the Clustal X (1.83) software, phylogenetic tree was constructed using MEGA 6.0. Its expression profile was performed by RT-PCR and qRT-PCR. Specific primers were designed to amplify the high specificity fragment, and then the PCR product was ligated to the PET-28 vector, which was transformed into Rosetta E.coli. The recombinant protein was induced by IPTG and purified by Ni⁺ affinity chromatography. Finally, the immune response was challenged by E. coli. Its relative mRNA level was detected by qRT-PCR.【Result】BmCat L was clustered on nscaf2860 which was located on chromosome 10, and the gene number in silkDB was BGIBMGA006893. Full length of its ORF is 687 bp, encoding 228 amino acids, there is a conservative Pept_C1 domain structure. The molecular weight and isoelectric point of the deduced protein is 26.132 kD and 4.57, respectively. Phylogenetic analysis showed that it is close to its homologous protein from Spodoptera frugiperda and Danaus plexippus. RT-PCR results showed that BmCat L was specifically and highly expressed in haemocyte. The recombinant protein of BmCat L was produced and purified, and finally verified by His-antibody. The immune challenge experiments results revealed the relationship between Cathepsin L gene and its immune system in B. mori.【Conclusion】 The Bmcat L cDNA was cloned. It was specific highly expressed in haemocytes. The recombinant proteins were obtained through prokaryotic expression and protein purification. More importantly, the expression of BmCat L was significantly increased after treating with E. coli in haemocytes. It is speculated that BmCat L might be involved in the immune response of B. mori.

Key words: BmCathepsin L, Bombyx mori, gene cloning, prokaryotic expression, protein purification, immune challenge

潘光照，张奎，李重阳，赵羽卒，申利，徐曼，苏晶晶，林西，崔红娟. 家蚕组织蛋白酶L（BmCathepsin L）基因鉴定、表达及其功能分析[J]. 中国农业科学, 2017, 50(16): 3236-3246.

PAN GuangZhao, ZHANG Kui, LI ChongYang, ZHAO YuZu, SHEN Li, XU Man, SU JingJing, LIN Xi, CUI HongJuan. Identification, Expression, and Functional Analysis of Cathepsin L in Silkworm (Bombyx mori)[J]. Scientia Agricultura Sinica, 2017, 50(16): 3236-3246.

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